Capillary electrophoresis using replaceable gels

ABSTRACT

The filing of an internally coated capillary with a gel in its polymerized state without damaging the gel. The coating prevents bonding of the gel to the inside of the capillary. The gel comprises up to 6% acrylamide and 0-5% crosslinker. The gel can be advantageously and conveniently used in automated electrophoresis systems for automatic replacement of spent gel.

This is a continuation of application Ser. No. 07/647,071, filed Jan.29, 1991 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to capillary gel electrophoresis, and moreparticularly to refilling capillaries using a polymerized gel.

2. Description of Related Art

Electrophoresis is one of the most widely used separation techniques inthe biologically related sciences. Molecular species such as peptides,proteins, and oligonucleotides (analytes) are separated by causing themto migrate at different rates in a separation medium under the influenceof an electric field. The separation medium can be a buffer solution, ora low to moderate concentration of an appropriate gelling agent such asagarose or polyacrylamide. When gel separation medium is used,separation of analytes is partly based on their molecular sizes as theanalytes are sieved by the gel matrix. Smaller molecules move relativelymore quickly than larger ones through a gel of a given pore size whichdepends in part on the concentration of the polymer in the gel.

U.S. Pat. Nos. 4,865,706 and 4,865,707 to Barry L. Karger and AharonsCohen describe gel compositions suitable for capillary electrophoresis.A fused silica capillary having inner diameter in the order of 75 μm isfirst filled with a mixture of acrylamide monomer and other ingredientsand polymerization is then allowed to go to completion in the capillary.The time taken to complete polymerization is a minimum of one hour. Thepolymerized gel has a limited storage life. Also, performance of the geldeteriorates after a period of use. This may be due to gradualaccumulation of macromolecules in the gel matrix after repeated runs.The applied electric field may cause disintegration of the polymermaterial after repeated use. In the past, the gel-filled capillarycolumns have to be discarded after their useful life.

The gel columns have heretofore been used in laboratory set-upsinvolving many manual steps, e.g. placement of buffer containers withrespect to the ends of the gel column, etc. Also spent gel column has tobe manually replaced by a new gel column. To take advantage ofautomation in carrying out electrophoresis, due considerations should begiven to eliminate as many of the manual steps in the design of anautomated electrophoresis system.

SUMMARY OF THE INVENTION

The present invention is directed to filling an internally coatedcapillary using a gel which is polymerized before filling the capillary.The internal coating on the capillary walls prevents bonding of the gelto the capillary walls. The gel is of a composition which allows it tofill the capillary in its polymerized state without damage to the geland to be removed from the capillary after the gel has expended itsuseful life. The capillary can then be refilled with fresh gel. This canbe handled automatically by an automated capillary electrophoresis

In the illustrated embodiment, the formulation of the gel comprises upto 6% of acrylamide monomer and buffer In addition, optional amounts ofcrosslinker, catalyst, initiator, urea, and other additives may be addedto adjust for the desired pore size, separation efficiency and life ofthe gel. The composition results in a gel of a consistency which can beforced into and out of the internally coated capillary without damagingthe gel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic layout of an automated electrophoresis system.

FIG. 2 is an electropherogram representing the results ofelectrophoresis using a gel injected into the capillary in a polymerizedstate according to the present invention.

FIG. 3 is an electropherogram representing the results ofelectrophoresis using the same gel by but which has been polymerized inthe capillary.

DETAILED DESCRIPTION OF THE INVENTION

The following description is of the best presently contemplated mode ofcarrying out the invention. This description is made for the purpose ofillustrating the general principles of the invention and should not betaken in a limiting sense. The scope of the invention is best determinedby reference to the appended claims.

The present invention can be used advantageously in conjunction with anautomated capillary electrophoresis systems, such as the P/ACE™ 2000electrophoresis system introduced by Beckman Instruments, Inc. Saidsystem is schematically shown in FIG. 1. The details of the system havebeen described in copending U.S. patent applications Ser. No.07/614,059; 07/542,673 and 07/187,760 commonly assigned to the assigneeof the present invention and are incorporated by reference herein. Inthat system, the capillary column 10 is encased in a cartridge 12 whichis supported to allow the ends of the capillary to access vials 14 and16 of electrolyte or sample solutions. Capillary referred herein meanstubing having an inside diameter typically less than 1000 μm and moretypically less than 300 μm. A detector 13 is provided to detectseparated species. The vials are carried on carousels 18 and 20 whichare rotated to position selected vials at the ends of the capillary. Aselected solution can be forced into the capillary by submerging one endof the capillary into the vial and pressurizing the vial by means notshown (for details see copending applications). According to the presentinvention, the polymerized gel can be contained in a vial on thecarousel and forced into an internally coated fused silica capillary inthe same manner as is done with solution. The capillary walls are coatedwith a material (e.g. 50% phenyl and 50% methyl, or cynopropyl) toprevent bonding of the gel to the walls. Since the instrument has abuilt-in rinse mode which runs a rinsing solution (typically a buffer orelectrolyte solution) through the capillary to clean the capillary, arinsing solution can thus be used to wash out the spent gel. Thecapillary 10 can then be refilled with fresh gel. AlternativeAlternatively, instead of using a rinse solution, a supply of fresh gelcan be used to displace the spent gel thus flushing and refilling thecapillary in a single step. The above steps can be carried outautomatically under control of microprocessor 30 programmed by the user.It can be seen that it would not be necessary to replace the entire gelcapillary column 10 to replace the gel which would involve removingcartridge from the system and removing the column from the cartridge 12.

The P/ACE™ system also has a sample injection mode which injects samplefrom a vial into one end of the capillary by either electromigration orpressure injection. Electrodes 26 and 28 are provided to apply therequired high voltage (in the order of several hundred volts per cm ofcapillary) from voltage supply 29 for electromigration injection as wellas for carrying out electrophoresis. Electrophoresis is performed withthe two ends of the capillary dipped into electrolyte containing vials.The electrolyte can be in the form of buffer solution similar to thebuffer the gel is made up of, or in the form of gel (i.e. a gel buffersystem). In the latter case, the gel in the capillary can be replacedwithout having to position another vial.

The basic composition of the refillable gel is up to 6% acrylamidemonomer dissolved in the appropriate buffer solution (usually 100 mMTRIS-borate of pH about 8.5). The acrylamide can be cross-linked with 0to 5% of methylenebisacrylamide (“BIS”). 7M urea, hydrophilic polymeradditives (e.g. polyethyleneglycol (“PEG”), an appropriate amount ofcatalyst (e.g. tetramethyleneethylenediamine (“TEMED”) and initiator(e.g. ammonium persulfate) and other additives may be added to obtain agel having the desired pore size, separation efficiency and life span.The steps for preparing the buffer and the gel are conventional and wellknown to one skilled in the art. Generally, the composition is allowedto polymerize overnight and the polymerized gel can be dialyzed orelectrodialyzed against the gel buffer in order to remove the remainingammonium persulfate, TEMED and the non-polymerized acrylamide andBIS-monomers if necessary. The coated inner surface of the fused silicacapillary can be treated before the first filling of the gel. Thesurface can be treated by using 100% solution ofmethacryloxypropyltrimethoxysilane for 1 hour at 50° C. A dilutedsolution (diluted with methanol) may also be used. The silane is for“neutralizing” any hydroxide ions remaining on the capillary walls as aresult of exposed silica due to slight imperfection in the coating. Thepresence of hydroxide ions is undesirable for some applications as itincreases electroendoosmosis.

The capillary can be refilled with fresh gel of the same or differentcomposition right after the previously spent gel has been removed fromthe capillary. Unlike the prior art gel columns where polymerizationtakes place in the capillary, it will not be necessary to wait forpolymerization to take place in the present invention once the capillaryhas been filled with polymerized gel. This complements the automatedfeatures of the automated electrophoresis system and eliminates waitingtime for changing of gel columns.

Up to 6% acrylamide without crosslinker, or up to 2% acrylamide +5%crosslinker, PEG of molecular weight up to 35,000 at concentration of upto 1% can be used is additive. Gels having high concentrations ofpolyacrylamide and crosslinker are too brittle to be able to be forcedin their polymerized state to fill the capillary without damaging thegel. However, it is believed that gel having acrylamide greater than 6%may also maintain its integrity under special injection conditions.Since prior art gel-filled capillary columns do not have internalcoating which prevents bonding of the gel to the capillary, the spentgel cannot be pushed out of the capillary effectively as the gel bondsto the capillary walls. It is noted that at concentration above 6% ofacrylamide without crosslinker, an appropriate increase of PEGconcentration and molecular weight is necessary to maintain therefillable property of the gel i.e. viscosity. Similarly, it has beenfound that in a composition having 5% BIS monomer and more than 2%acrylamide monomer, the PEG concentration and molecular weight should beincreased.

Examples of specific compositions of the refillable gels according tothe present invention which performance have been found to be comparableto prior art non-refillable gels are given below. The following examplesare offered for illustrative purposes only, and are intended neither todefine nor limit the invention in any manner.

EXAMPLE 1

100 Mm TRIS

100 Mm Boric Acid

3% T

0.5% C

2 Mm EDTA (for separation of polynucleotides)

8.35 pH

EXAMPLE 2

100 Mm TRIS

100 Mm Boric Acid

1% T

5% C

2 Mm EDTA (for separation of polynucleotides)

8.35 pH

In both examples above, the acrylamide and crosslinker concentrationsare expressed in %T and %C to characterize the gels. The definitions of%T and %C are as follows:${\% T} = {\frac{{{mg}\quad {acrylamide}} + {{mg}\quad {crosslinker}}}{{ml}\quad {buffer}\quad {volume}} \times 100}$${\% C} = {\frac{{mg}\quad {crosslinker}}{{{mg}\quad {acrylamide}} + {{mg}\quad {crosslinker}}} \times 100}$

The total amount as well is the ratio of acrylamide and crosslinkerdetermine the pore size and the pore distribution of a polyacrylamidegel. In the examples given, there is 2.985% acrylamide and 0.015%crosslinker (BIS) in Example 1 and 0.95% acrylamide and 0.05%crosslinker in Example 2. The crosslinker may be omitted in Example 1 ifdesired.

The results of electrophoresis using the gel of Example 1 is representedby the electropherogram in FIG. 2. The sample undergoing electrophoresisis φ X-174 RF DNA-Hae III Digest mixture. The capillary column has aninner coating (e.g. OV-17) for preventing bonding of the gel to thecapillary walls. The dimension of the capillary is 100 μ I.D., 47 cmtotal length with 40 cm effective length. The gel was polymerized andthen injected into the capillary by using the rinse mode of the P/ACE™2000 system. The sample is of 1 mg/ml concentration and is injected byelectromigration into the gel column by applying 5 KV for 2 seconds. Theelectrophoresis is carried out under 12 KV and 33 μA.

The results in FIG. 2 can be compared to the results shown in FIG. 3which represents the electropherogram of the same sample separated in agel column having the same composition but which has the gel polymerizedin the column. Sample injection and run parameters are the same. It isseen that the resolution of the peaks in the two electropherograms arequite similar. While the amplitudes between the two results differsomewhat, it is however not as much a concern as peak resolution forpurposes of gel electrophoresis analysis as it is not a quantitativeanalysis.

Accordingly, it has been demonstrated that gel columns that have beenrefilled according to the present invention provide substantially thesame separation efficiency, power and resolution as compared to the samegel polymerized in the capillary. The gel is not damaged as it is beingforced into the coated capillary in its polymerized state therebymaintaining its separation performance. The refillable gel can be usedadvantageously in automated electrophoresis systems in which the task ofreplacing fresh gel can be handled automatically. A series of runsincluding changing of gel between runs can be programmed to beper-formed automatically without user intervention.

While the invention has been described with respect to the preferredembodiments in accordance therewith, it will be apparent to thoseskilled in the art that various modifications and improvements may bemade without departing from the scope and spirit of the invention.Accordingly, it is to be understood that the invention is not to belimited by the specific described embodiments, but only by the scope ofthe appended claims.

I claim:
 1. A method of forming a gel filled capillary useful forcapillary electrophoresis comprising the steps of: providing a capillaryproviding polymerized gel; and filling the capillary with polymerizedgel.
 2. A method of as in claim 1A method of forming a gel filledcapillary useful for capillary electrophoresis comprising: providing acapillary; providing a polymerized gel; and filling the capillary withthe polymerized gel by an application of force, wherein the capillaryprovided has a coating on the inside of the capillary which preventsbonding of the gel to the coating.
 3. A method as in claim 2 wherein themonomer is acrylamide and the gel has composition comprising up to 6%acrylamide and 0-5% crosslinker.
 4. A method as in claim 3 wherein thecrosslinker comprises methylenebisacrylamide.
 5. A method as in claim 1wherein the polymerized gel is provided by providing polymerizablemonomer and polymerizing the monomer into gel.
 6. A method of separatinga sample into its molecular species comprising the steps of: providing acapillary; providing polymerized gel useful for electrophoresis; fillingthe capillary with the polymerized gel; introducing a sample into thefilled capillary; and performing electrophoresis on the sample toseparate the sample into its molecular species.
 7. A method as in claim6 further comprising the steps: A method of separating a sample into itsmolecular species comprising: providing a capillary; providing apolymerized gel useful for electrophoresis; filling the capillary withthe polymerized gel by an application of force; introducing a sampleinto the filled capillary; performing electrophoresis on the sample toseparate the sample into its molecular species; removing the gel fromthe capillary after electrophoresis; and refilling the capillary withpolymerized gel.
 8. A method as in claim 7A method of separating asample into its molecular species comprising: providing a capillary;providing a polymerized gel useful for electrophoresis; filling thecapillary with the polymerized gel by an application of force;introducing a sample into the filled capillary; performingelectrophoresis on the sample to separate the sample into its molecularspecies; removing the gel from the capillary after electrophoresis; andrefilling the capillary with polymerized gel; wherein the removing andrefilling step are performed at the same time by using polymerized gelto displace the gel originally in the capillary.
 9. A method as in claim6 7whereby the capillary provided has a coating on the inside of thecapillary which prevents bonding of the gel to the coating.
 10. A methodas in claim 9 wherein the gel comprises up to 6% acrylamideand 0-5%crosslinker .
 11. A method as in claim 6of separating a sample into itsmolecular species comprising: providing a capillary; providing apolymerized gel useful for electrophoresis; filling the capillary withthe polymerized gel by an application of force; introducing a sampleinto the filled capillary; performing electrophoresis on the sample toseparate the sample into its molecular species wherein the polymerizedgel is provided by providing polymerizable monomer and polymerizing themonomer into gel; removing the gel from the capillary afterelectrophoresis; and refilling the capillary with polymerized gel.
 12. Amethod as in claim 11 wherein the monomer is acrylamide and the gel hasa composition comprising up to 6% acrylamide.
 13. A method of separatinga sample into its molecular species comprising: providing a capillary;providing a polymerized gel useful for electrophoresis; filling thecapillary with the polymerized gel by an application of force;introducing a sample into the filled capillary; performingelectrophoresis on the sample to separate the sample into its molecularspecies; removing the gel from the capillary after electrophoresis;refilling the capillary with polymerized gel; and automaticallycontrolling operation of an apparatus wherein the polymerized gel andsample are contained in receptacles.
 14. A method of separating a sampleinto its molecular species comprising: providing a capillary; providinga polymerized gel useful for electrophoresis; filling the capillary withthe polymerized gel by an application of force; introducing a sampleinto the filled capillary; performing electrophoresis on the sample toseparate the sample into its molecular species; removing the gel fromthe capillary after electrophoresis; refilling the capillary withpolymerized gel; and automatically controlling operation of an apparatuswherein the polymerized gel and sample are contained in receptacles, andwherein the apparatus further comprises a carrier for supporting thereceptacles and means for turning the carrier to position the carrier tothe respective receptacle in flow communication with the capillary. 15.The method of claim 2, wherein the polymerized gel comprisescrosslinker.
 16. The method of claim 7, wherein the removing stepcomprises running a solution into the capillary to remove spent gel. 17.The method of claim 7, wherein the removing and refilling steps areperformed at the same time by using polymerized gel to displace the geloriginally in the capillary.
 18. The method of claim 8, wherein theremoving step comprises running a solution into the capillary to removespent gel.
 19. The method of claim 11, wherein the removing stepcomprises running a solution into the capillary to remove spent gel. 20.The method of claim 11, wherein the removing and refilling steps areperformed at the same time by using polymerized gel to displace the geloriginally in the capillary.
 21. The method of claim 10, furthercomprising up to 5% crosslinker.
 22. The method of claim 12, furthercomprising up to 5% crosslinker.